PowerPoint Presentation

Mass Spectrometry: Methods & Theory1Proteomics ToolsMolecular Biology ToolsSeparation & Display ToolsProtein Identification ToolsProtein Structure ToolsMass Spectrometry NeedsIonization-how the protein is injected in to the MS machineSeparation-Mass and Charge is determinedActivation-protein are broken into smaller fragments (peptides/AAs)Mass Determination-m/z ratios are determined for the ionized protein fragments/peptidesProtein Identification2D-GE + MALDI-MSPeptide Mass Fingerprinting (PMF)

2D-GE + MS-MSMS Peptide Sequencing/Fragment Ion Searching

Multidimensional LC + MS-MSICAT Methods (isotope labelling)MudPIT (Multidimensional Protein Ident. Tech.)

1D-GE + LC + MS-MS

De Novo Peptide Sequencing

Mass Spectrometry (MS)Introduce sample to the instrumentGenerate ions in the gas phaseSeparate ions on the basis of differences in m/z with a mass analyzer Detect ions

How does a mass spectrometer work?Ionization methodMALDIElectrospray(Proteins must be charged and dry)Mass analyzerMALDI-TOFMW Triple QuadrapoleAA seqMALDI-QqTOFAA seq and MWQqTOFAA seq and protein modif.

Create ionsSeparate ionsDetect ionsMass spectrumDatabase analysisGeneralized Protein Identification by MSArtificial spectra builtArtificially trypsinatedDatabase of sequences(i.e. SwissProt)Spot removed from gelFragmented using trypsinSpectrum of fragments generatedMATCHLibrary

Methods for protein identificationMS PrinciplesDifferent elements can be uniquely identified by their mass

MS PrinciplesDifferent compounds can be uniquely identified by their massCH3CH2OHNOHHO-CH2--CH2CH-NH2COOHHOHOButorphanol L-dopa EthanolMW = 327.1 MW = 197.2 MW = 46.1Mass SpectrometryAnalytical method to measure the molecular or atomic weight of samples

Weighing proteinsA mass spectrometer creates charged particles (ions) from molecules.

Common way is to add or take away an ions:

NaCl + e- NaCl-NaCl NaCl+ + e-

It then analyzes those ions to provide information about the molecular weight of the compound and its chemical structure.

Mass SpectrometryFor small organic molecules the MW can be determined to within 5 ppm or 0.0005% which is sufficiently accurate to confirm the molecular formula from mass alone

For large biomolecules the MW can be determined within an accuracy of 0.01% (i.e. within 5 Da for a 50 kD protein)

Recall 1 dalton = 1 atomic mass unit (1 amu)MS HistoryJJ Thomson built MS prototype to measure m/z of electron, awarded Nobel Prize in 1906

MS concept first put into practice by Francis Aston, a physicist working in Cambridge England in 1919

Designed to measure mass of elements

Aston Awarded Nobel Prize in 1922

MS History1948-52 - Time of Flight (TOF) mass analyzers introduced

1955 - Quadrupole ion filters introduced by W. Paul, also invents the ion trap in 1983 (wins 1989 Nobel Prize)

1968 - Tandem mass spectrometer appears

Mass spectrometers are now one of the MOST POWERFUL ANALYTIC TOOLS IN CHEMISTRY

MS PrinciplesFind a way to charge an atom or molecule (ionization)

Place charged atom or molecule in a magnetic field or subject it to an electric field and measure its speed or radius of curvature relative to its mass-to-charge ratio (mass analyzer)

Detect ions using microchannel plate or photomultiplier tubeMass Spec PrinciplesIonizerSample+_Mass AnalyzerDetectorHow does a mass spectrometer work?Ionization methodMALDIElectrospray(Proteins must be charged and dry)Mass analyzerMALDI-TOFMW Triple QuadrapoleAA seqMALDI-QqTOFAA seq and MWQqTOFAA seq and protein modif.

Create ionsSeparate ionsDetect ionsMass spectrumDatabase analysisMass spectrometers

Time of flight (TOF) (MALDI)Measures the time required for ions to fly down the length of a chamber. Often combined with MALDI (MALDI-TOF) Detections from multiple laser bursts are averaged. Multiple laser

Tandem MS- MS/MS-separation and identification of compounds in complex mixtures- induce fragmentation and mass analyze the fragment ions. - Uses two or more mass analyzers/filters separated by a collision cell filled with Argon or Xenon

Different MS-MS configurationsQuadrupole-quadrupole (low energy)Magnetic sector-quadrupole (high)Quadrupole-time-of-flight (low energy)Time-of-flight-time-of-flight (low energy)Typical Mass Spectrometer

LC/LC-MS/MS-Tandem LC, Tandem MSTypical Mass SpectrumCharacterized by sharp, narrow peaks

X-axis position indicates the m/z ratio of a given ion (for singly charged ions this corresponds to the mass of the ion)

Height of peak indicates the relative abundance of a given ion (not reliable for quantitation)

Peak intensity indicates the ions ability to desorb or fly (some fly better than others)

m/z ratio:Molecular weight divided by the charge on this proteinAll proteins are sorted based on a mass to charge ratio (m/z)Typical Mass Spectrum

aspirinRelative Abundance120 m/z-for singly charged ion this is the massResolution & Resolving PowerWidth of peak indicates the resolution of the MS instrument

The better the resolution or resolving power, the better the instrument and the better the mass accuracy

Resolving power is defined as:M is the mass number of the observed mass (DM) is the difference between two masses that can be separated

DMMResolution in MS

Resolution in MS

QTOF783.455784.465785.475783.6Mass Spectrometer SchematicInletIon SourceMassFilterDetectorDataSystemHigh Vacuum SystemTurbo pumpsDiffusion pumpsRough pumpsRotary pumpsSample PlateTargetHPLCGCSolids probeMALDIESIIonSprayFABLSIMSEI/CITOFQuadrupoleIon TrapMag. SectorFTMSMicroch plateElectron Mult.Hybrid Detec.PCsUNIXMacDifferent Ionization MethodsElectron Impact (EI - Hard method)small molecules, 1-1000 Daltons, structure

Fast Atom Bombardment (FAB Semi-hard)peptides, sugars, up to 6000 Daltons

Electrospray Ionization (ESI - Soft)peptides, proteins, up to 200,000 Daltons

Matrix Assisted Laser Desorption (MALDI-Soft)peptides, proteins, DNA, up to 500 kDElectron Impact IonizationSample introduced into instrument by heating it until it evaporates

Gas phase sample is bombarded with electrons coming from rhenium or tungsten filament (energy = 70 eV)

Molecule is shattered into fragments (70 eV >> 5 eV bonds)

Fragments sent to mass analyzer

EI Fragmentation of CH3OHCH3OHCH3OH+CH3OHCH2O=H++ HCH3OH+ CH3+ OHCHO=H++ HCH2O=H+Why wouldnt Electron Impact be suitable for analyzing proteins?Why You Cant Use EI For Analyzing ProteinsEI shatters chemical bonds

Any given protein contains 20 different amino acids

EI would shatter the protein into not only into amino acids but also amino acid sub-fragments and even peptides of 2,3,4 amino acids

Result is 10,000s of different signals from a single protein -- too complex to analyzeSoft Ionization Methods337 nm UV laserMALDIcyano-hydroxycinnamic acidGold tip needleFluid (no salt)ESI+_Soft Ionization Soft ionization techniques keep the molecule of interest fully intact

Electro-spray ionization first conceived in 1960s by Malcolm Dole but put into practice in 1980s by John Fenn (Yale)

MALDI first introduced in 1985 by Franz Hillenkamp and Michael Karas (Frankfurt)

Made it possible to analyze large molecules via inexpensive mass analyzers such as quadrupole, ion trap and TOF

Ionization methodsElectrospray mass spectrometry (ESI-MS)Liquid containing analyte is forced through a steel capillary at high voltage to electrostatically disperse analyte. Charge imparted from rapidly evaporating liquid.

Matrix-assisted laser desorption ionization (MALDI)Analyte (protein) is mixed with large excess of matrix (small organic molecule)Irradiated with short pulse of laser light. Wavelength of laser is the same as absorbance max of matrix.

Electrospray IonizationSample dissolved in polar, volatile buffer (no salts) and pumped through a stainless steel capillary (70 - 150 mm) at a rate of 10-100 mL/min

Strong voltage (3-4 kV) applied at tip along with flow of nebulizing gas causes the sample to nebulize or aerosolize

Aerosol is directed through regions of higher vacuum until droplets evaporate to near atomic size (still carrying charges)Electrospray (Detail)

Electrospray IonizationCan be modified to nanospray system with flow < 1 mL/min

Very sensitive technique, requires less than a picomole of material

Strongly affected by salts & detergents

Positive ion mode measures (M + H)+ (add formic acid to solvent)

Negative ion mode measures (M - H)- (add ammonia to solvent)Positive or Negative Ion Mode?If the sample has functional groups that readily accept H+ (such as amide and amino groups found in peptides and proteins) then positive ion detection is used-PROTEINS

If a sample has functional groups that readily lose a proton (such as carboxylic acids and hydroxyls as found in nucleic acids and sugars) then negative ion detection is used-DNAMatrix-Assisted Laser Desorption Ionization337 nm UV laserMALDIcyano-hydroxycinnamic acidMALDISample is ionized by bombarding sample with laser light

Sample is mixed with a UV absorbant matrix (sinapinic acid for proteins, 4-hydroxycinnaminic acid for peptides)

Light wavelength matches that of absorbance maximum of matrix so that the matrix transfers some of its energy to the analyte (leads to ion sputtering)HT Spotting on a MALDI Plate

MALDI Ionization++++---++++----++AnalyteMatrixLaser+++Absorption of UV radiation by chromophoric matrix and ionization of matrix

Dissociation of matrix, phase change to super-compressed gas, charge transfer to analyte molecule

Expansion of matrix at supersonic velocity, analyte trapped in expanding matrix plume (explosion/popping)+++MALDIUnlike ESI, MALDI generates spectra that have just a singly charged ion

Positive mode generates ions of M + H

Negative mode generates ions of M - H

Generally more robust that ESI (tolerates salts and nonvolatile components)

Easier to use and maintain, capable of higher throughput

Requires 10 mL of 1 pmol/mL samplePrincipal for MALDI-TOF MASS

Principal for MALDI-TOF MASS

MALDI = SELDI337 nm UV laserMALDIcyano-hydroxycinnaminic acid

MALDI/SELDI Spectra

NormalTumorMassFilterMass Spectrometer SchematicInletIon SourceDetectorDataSystemHigh Vacuum SystemTurbo pumpsDiffusion pumpsRough pumpsRotary pumpsSample PlateTargetHPLCGCSolids probeMALDIESIIonSprayFABLSIMSEI/CITOFQuadrupoleIon TrapMag. SectorFTMSMicroch plateElectron Mult.Hybrid Detec.PCsUNIXMacDifferent Mass AnalyzersMagnetic Sector Analyzer (MSA)High resolution, exact mass, original MA

Quadrupole Analyzer (Q)Low (1 amu) resolution, fast, cheap

Time-of-Flight Analyzer (TOF)No upper m/z limit, high throughput

Ion Trap Mass Analyzer (QSTAR)Good resolution, all-in-one mass analyzer

Ion Cyclotron Resonance (FT-ICR)Highest resolution, exact mass, costlyDifferent Types of MSESI-QTOFElectrospray ionization source + quadrupole mass filter + time-of-flight mass analyzer

MALDI-QTOFMatrix-assisted laser desorption ionization + quadrupole + time-of-flight mass analyzer

Both separate by MW and AA seqDifferent Types of MSGC-MS - Gas Chromatography MSseparates volatile compounds in gas column and IDs by mass

LC-MS - Liquid Chromatography MSseparates delicate compounds in HPLC column and IDs by mass

MS-MS - Tandem Mass Spectrometryseparates compound fragments by magnetic field and IDs by mass

LC/LC-MS/MS-Tandem LC and Tandem MSSeparates by HPLC, IDs by mass and AA sequence

Magnetic Sector AnalyzerQuadrupole Mass AnalyzerA quadrupole mass filter consists of four parallel metal rods with different charges

Two opposite rods have an applied + potential and the other two rods have a - potential

The applied voltages affect the trajectory of ions traveling down the flight path

For given dc and ac voltages, only ions of a certain mass-to-charge ratio pass through the quadrupole filter and all other ions are thrown out of their original path Quadrupole Mass Analyzer

Q-TOF Mass AnalyzerTOFNANOSPRAY TIPIONSOURCEHEXAPOLECOLLISIONCELLHEXAPOLEHEXAPOLEQUADRUPOLEMCPDETECTORREFLECTRONSKIMMERPUSHERMass Spec Equation (TOF)mz2Vt2=m = mass of ionL = drift tube lengthz = charge of iont = time of travelV = voltageL2Ion Trap Mass AnalyzerIon traps are ion trapping devices that make use of a three-dimensional quadrupole field to trap and mass-analyze ions

invented by Wolfgang Paul (Nobel Prize1989)

Offer good mass resolving power

FT-ICRFourier-transform ion cyclotron resonanceUses powerful magnet (5-10 Tesla) to create a miniature cyclotron

Originally developed in Canada (UBC) by A.G. Marshal in 1974

FT approach allows many ion masses to be determined simultaneously (efficient)

Has higher mass resolution than any other MS analyzer available

FT-Ion Cyclotron Analzyer

Current Mass Spec TechnologiesProteome profiling/separation2D SDS PAGE - identify proteins2-D LC/LC - high throughput analysis of lysates(LC = Liquid Chromatography)2-D LC/MS (MS= Mass spectrometry)

Protein identificationPeptide mass fingerprintTandem Mass Spectrometry (MS/MS)

Quantative proteomicsICAT (isotope-coded affinity tag)ITRAQ 2D - LC/LC

Study protein complexes without gel electrophoresisPeptides all bind to cation exchange columnPeptides are separated by hydrophobicity on reverse phase columnSuccessive elution with increasing salt gradients separates peptides by chargeComplex mixture is simplified prior to MS/MS by 2D LC(trypsin)

2D - LC/MSPeptide Mass Fingerprinting (PMF)

Peptide Mass FingerprintingUsed to identify protein spots on gels or protein peaks from an HPLC run

Depends of the fact that if a peptide is cut up or fragmented in a known way, the resulting fragments (and resulting masses) are unique enough to identify the protein

Requires a database of known sequences

Uses software to compare observed masses with masses calculated from databasePrinciples of Fingerprinting>Protein 1acedfhsakdfqeasdfpkivtmeeewendadnfekqwfe

>Protein 2acekdfhsadfqeasdfpkivtmeeewenkdadnfeqwfe

>Protein 3acedfhsadfqekasdfpkivtmeeewendakdnfeqwfeSequence Mass (M+H) Tryptic Fragments4842.05

4842.05

4842.05 acedfhsakdfgeasdfpkivtmeeewendadnfekgwfe

acekdfhsadfgeasdfpkivtmeeewenkdadnfeqwfe

acedfhsadfgekasdfpkivtmeeewendakdnfegwfe

Principles of Fingerprinting>Protein 1acedfhsakdfqeasdfpkivtmeeewendadnfekqwfe

>Protein 2acekdfhsadfqeasdfpkivtmeeewenkdadnfeqwfe

>Protein 3acedfhsadfqekasdfpkivtmeeewendakdnfeqwfeSequence Mass (M+H) Mass Spectrum4842.05

4842.05

4842.05 Predicting Peptide Cleavageshttp://ca.expasy.org/tools/peptidecutter/

http://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#TrypsProtease Cleavage RulesTrypsinXXX[KR]--[!P]XXXChymotrypsinXX[FYW]--[!P]XXXLys CXXXXXK-- XXXXXAsp N endoXXXXXD-- XXXXXCNBrXXXXXM--XXXXXK-Lysine, R-Arginine, F-Phenylalanine, Y-Tyrosine, W-Tryptophan,D-Aspartic Acid, M-Methionine, P-ProlineSometimes inhibition occursWhy Trypsin?Robust, stable enzymeWorks over a range of pH values & Temp.Quite specific and consistent in cleavageCuts frequently to produce ideal MW peptidesInexpensive, easily available/purifiedDoes produce autolysis peaks (which can be used in MS calibrations)1045.56, 1106.03, 1126.03, 1940.94, 2211.10, 2225.12, 2283.18, 2299.18Digest with specific protease>RBME00320 Contig0311_1089618_1091255 EC-mopA 60 KDa chaperonin GroELMAAKDVKFGR TAREKMLRGV DILADAVKVT LGPKGRNVVI EKSFGAPRIT KDGVSVAKEV ELEDKFENMG AQMLREVASK TNDTAGDGTT TATVLGQAIV QEGAKAVAAG MNPMDLKRGI DLAVNEVVAE LLKKAKKINT SEEVAQVGTI SANGEAEIGK MIAEAMQKVG NEGVITVEEA KTAETELEVV EGMQFDRGYL SPYFVTNPEK MVADLEDAYI LLHEKKLSNL QALLPVLEAV VQTSKPLLII AEDVEGEALA TLVVNKLRGG LKIAAVKAPG FGDCRKAMLE DIAILTGGQV ISEDLGIKLE SVTLDMLGRA KKVSISKENT TIVDGAGQKA EIDARVGQIK QQIEETTSDY DREKLQERLA KLAGGVAVIR VGGATEVEVK EKKDRVDDAL NATRAAVEEG IVAGGGTALL RASTKITAKG VNADQEAGIN IVRRAIQAPA RQITTNAGEE ASVIVGKILE NTSETFGYNT ANGEYGDLIS LGIVDPVKVV RTALQNAASV AGLLITTEAM IAELPKKDAA PAGMPGGMGG MGGMDF546 aa60 kDa; 57 461 Da pI = 4.75Digest with specific proteaseTrypsin yields 47 peptides (theoretically)501.3533.3544.3545.3614.4634.3674.3675.4701.4726.4822.4855.5861.4879.4921.5953.4974.5988.51000.61196.61217.61228.51232.61233.71249.61249.61344.71455.81484.61514.81582.91583.91616.81726.71759.91775.91790.61853.91869.92286.22302.22317.22419.22526.42542.43329.64211.4

Peptide masses in Da:http://us.expasy.org/tools/peptide-mass.htmlDigest with trypsinIn practice.......see far fewer by mass spec- possibly incomplete digest (we allow 1 miss)- lose peptides during each manipulationwashes during digestionwashes during cleanup stepsome peptides will not ionize wellsome signals (peaks) are poorlow intensity; lack resolutionWhat Are Missed Cleavages?>Protein 1acedfhsakdfqeasdfpkivtmeeewendadnfekqwfeSequenceTryptic Fragments (no missed cleavage)acedfhsak (1007.4251) dfgeasdfpk (1183.5266) ivtmeeewendadnfek (2098.8909) gwfe (609.2667)

Tryptic Fragments (1 missed cleavage)acedfhsak (1007.4251) dfgeasdfpk (1183.5266) ivtmeeewendadnfek 2098.8909) gwfe (609.2667)acedfhsakdfgeasdfpk (2171.9338)ivtmeeewendadnfekgwfe (2689.1398)dfgeasdfpkivtmeeewendadnfek (3263.2997)Calculating Peptide MassesSum the monoisotopic residue massesMonoisotopic Mass: the sum of the exact or accurate masses of the lightest stable isotope of the atoms in a moleculeAdd mass of H2O (18.01056)Add mass of H+ (1.00785 to get M+H)If Met is oxidized add 15.99491If Cys has acrylamide adduct add 71.0371If Cys is iodoacetylated add 58.0071Other modifications are listed athttp://prowl.rockefeller.edu/aainfo/deltamassv2.html

1H-1.007828503 amu 12C-122H-2.014017780 amu13C-13.00335, 14C-14.00324Masses in MS

Monoisotopic mass is the mass determined using the masses of the most abundant isotopes

Average mass is the abundance weighted mass of all isotopic componentsMass Calculation (Glycine)NH2CH2COOH R1NHCH2COR3 Amino acidResidueMonoisotopic Mass1H = 1.00782512C = 12.0000014N = 14.0030716O = 15.99491Glycine Amino Acid Mass5xH + 2xC + 2xO + 1xN= 75.032015 amuGlycine Residue Mass3xH + 2xC + 1xO + 1xN=57.021455 amuAmino Acid Residue MassesGlycine57.02147Alanine71.03712Serine87.03203Proline97.05277Valine99.06842Threonine101.04768Cysteine103.00919Isoleucine113.08407Leucine113.08407Asparagine114.04293Aspartic acid115.02695Glutamine128.05858Lysine128.09497Glutamic acid129.0426Methionine131.04049Histidine137.05891Phenylalanine147.06842Arginine156.10112Tyrosine163.06333Tryptophan186.07932Monoisotopic MassAmino Acid Residue MassesGlycine57.0520Alanine71.0788Serine87.0782Proline97.1167Valine99.1326Threonine101.1051Cysteine103.1448Isoleucine113.1595Leucine113.1595Asparagine114.1039Aspartic acid115.0886Glutamine128.1308Lysine128.1742Glutamic acid129.1155Methionine131.1986Histidine137.1412Phenylalanine147.1766Arginine156.1876Tyrosine163.1760Tryptophan186.2133Average MassPreparing a Peptide Mass Fingerprint DatabaseTake a protein sequence database (Swiss-Prot or nr-GenBank)

Determine cleavage sites and identify resulting peptides for each protein entry

Calculate the mass (M+H) for each peptideSort the masses from lowest to highest

Have a pointer for each calculated mass to each protein accession number in databankBuilding A PMF Database>P12345acedfhsakdfqeasdfpkivtmeeewendadnfekqwfe

>P21234acekdfhsadfqeasdfpkivtmeeewenkdadnfeqwfe

>P89212acedfhsadfqekasdfpkivtmeeewendakdnfeqwfeSequence DBCalc. Tryptic Frags Mass Listacedfhsakdfgeasdfpkivtmeeewendadnfekgwfe

acekdfhsadfgeasdfpkivtmeeewenkdadnfeqwfe

acedfhsadfgekasdfpkivtmeeewendakdnfegwfe

450.2017 (P21234) 609.2667 (P12345) 664.3300 (P89212) 1007.4251 (P12345)1114.4416 (P89212)1183.5266 (P12345)1300.5116 (P21234) 1407.6462 (P21234)1526.6211 (P89212)1593.7101 (P89212) 1740.7501 (P21234) 2098.8909 (P12345)

The Fingerprint (PMF) AlgorithmTake a mass spectrum of a trypsin-cleaved protein (from gel or HPLC peak)

Identify as many masses as possible in spectrum (avoid autolysis peaks of trypsin)

Compare query masses with database masses and calculate # of matches or matching score (based on length and mass difference)

Rank hits and return top scoring entry this is the protein of interestQuery (MALDI) Spectrum500 1000 1500 2000 25006982098119910076094502211 (trp)1940 (trp)Query vs. DatabaseQuery Masses Database Mass List Results450.2017 (P21234) 609.2667 (P12345) 664.3300 (P89212) 1007.4251 (P12345)1114.4416 (P89212)1183.5266 (P12345)1300.5116 (P21234) 1407.6462 (P21234)1526.6211 (P89212)1593.7101 (P89212) 1740.7501 (P21234) 2098.8909 (P12345)

450.2201609.3667698.31001007.53911199.49162098.99092 Unknown masses1 hit on P212343 hits on P12345

Conclude the queryprotein is P12345Database search

theoreticalexperimentalMascotProtein IDPeptIdent (ExPasy)Mascot (Matrix Science)MS-Fit (Prospector; UCSF)ProFound (Proteometrics)MOWSE (HGMP) Human Genome Mapping Project

What You Need To Do PMFA list of query masses (as many as possible)

Protease(s) used or cleavage reagents

Databases to search (SWProt, Organism)

Estimated mass and pI of protein spot (opt) Cysteine (or other) modifications

Minimum number of hits for significance

Mass tolerance (100 ppm = 1000.0 0.1 Da)

A PMF website (Prowl, ProFound, Mascot, etc.)PMF on the WebProFoundhttp://129.85.19.192/profound_bin/WebProFound.exeMOWSEhttp://srs.hgmp.mrc.ac.uk/cgi-bin/mowsePeptideSearchhttp://www.narrador.embl-heidelberg.de/GroupPages/Homepage.htmlMascotwww.matrixscience.comPeptIdenthttp://us.expasy.org/tools/peptident.html

ProFoundProFound Results

MOWSE

PeptIdent

MASCOT

Mascot ScoringThe statistics of peptide fragment matching in MS (or PMF) is very similar to the statistics used in BLAST

The scoring probability follows an extreme value distribution

High scoring segment pairs (in BLAST) are analogous to high scoring mass matches in Mascot

Mascot scoring is much more robust than arbitrary match cutoffs (like % ID) Extreme Value Distributionit is the limit distribution of the maxima of a sequence of independent and identically distributed random variables. Because of this, the EVD is used as an approximation to model the maxima of long (finite) sequences of random variables.

P(x) = 1 - e-e-x Scores greater than 72 are significantMASCOT

Mascot/Mowse ScoringThe Mascot Score is given as S = -10*Log(P), where P is the probability that the observed match is a random event

Try to aim for probabilities where P